CN109705841B - Gold nanocluster with transferrin as template and preparation method and application thereof - Google Patents

Abstract

The invention provides a gold nanocluster taking transferrin as a template and a preparation method and application thereof. The preparation method of the nanocluster comprises the following steps: respectively preparing HAuCl₄And Tf in a molar ratio of 20-60:1, stirring at room temperature for 2-5 min, adjusting the pH of the mixed solution to 12 with NaOH solution, placing the solution in a microwave reactor, sealing, and reacting at 70-90 ℃ for at least 50min to obtain AuNCs @ Tf with strong red fluorescence emission. The nanocluster can be used for detecting copper ions and applied to copper ion detection test paper to enable the detection of the copper ions to be visual. The nanoclusters of the invention can also be used for detecting glutathione, and cell experiments show that the nanoclusters can target lysosomes to identify cancer cells. The nanoclusters of the present invention may also be used for encryption and decryption of fingerprint information.

Description

Gold nanocluster with transferrin as template and preparation method and application thereof

Technical Field

The invention relates to a fluorescence detection material, in particular to a red fluorescence gold nanocluster taking transferrin as a template and a preparation method and application thereof.

Background

Copper ion (Cu)²⁺) Is one of the trace elements in human beings and other mammals, and plays a key role in our health. Cu²⁺Can cause intestinal disorders and liver or kidney damage as well as several neurodegenerative diseases, such as alzheimer's disease, parkinson's disease and wilson's disease. In addition, excessive industrial emissions can also cause environmental pollution. Therefore, a sensitive method for detecting copper ions is found, and the method has important value for detecting the copper ion pollution of food and environment.

Glutathione (GSH) is a tripeptide containing sulfhydryl and gamma-amide bonds that play a key role in many physiological and pathological processes. Every cell of the body contains essentially glutathione, which is composed mainly of glutamate, cysteine and glycine. Normal levels of GSH (cytoplasm (2-10mM) and plasma (2-20. mu.M)) have the effects of maintaining a normal immune system, resisting oxidation and overall detoxification. However, excess GSH is associated with diseases including cancer, leukocyte loss, diabetes, and human immunodeficiency virus. On the other hand, GSH deficiency has been reported to lead to aging, liver disease and neurodegenerative disease. Interestingly, previously reported tumor tissues have more reducing environment than normal tissues, and their GSH concentrations are several times higher than normal tissues. Therefore, it is important to detect GSH levels.

Currently, there are many techniques for measuring GSH, including high performance liquid chromatography, surface enhanced raman scattering, fluorescence spectroscopy, enzyme-linked immunosorbent assays, electrochemical voltammetry, uv-visualization, and capillary electrophoresis. However, these techniques are often expensive, complex, time consuming, and impractical for biological system applications. Recently, attention has been paid to the development of fluorescent probes for GSH detection, which have the advantages of simple operation, mild reaction conditions, high sensitivity and fast response. Disappointingly, the similar structures and properties of cysteine (Cys), homocysteine (Hcy) and Glutathione (GSH) in mammalian cells present several challenges for the selective and discriminatory detection of each individual thiol. Many fluorescent probes, particularly fluorescent dyes, have been explored to date. However, most organic dyes have narrow excitation spectra and are poorly photostable, cytotoxic and environmental-risk, which limits their use. In addition, carbon quantum dot fluorescent probes (MnO) have been developed₂CDs) for the identification of GSH. Unfortunately, quantum dots containing heavy metals are very harsh and potentially cytotoxic to reaction conditions. Gold nanoclusters have large stokes shifts, good biocompatibility, and show great potential for in vitro and in vivo imaging compared to these materials.

Based on the method, the gold nanocluster emitting strong red fluorescence is obtained by reducing and coating tetrachloroauric acid with transferrin under the microwave condition, and the fluorescence property is utilized to detect copper ions, detect GSH, target lysosome to identify cancer cells and encrypt and decrypt fingerprint information.

Disclosure of Invention

The invention aims to provide a preparation method of a red fluorescent gold nanocluster taking transferrin as a template, which is simple and quick; the prepared nanoclusters can detect copper ions, detect GSH, target lysosomes to identify cancer cells and encrypt and decrypt fingerprint information.

The invention provides a preparation method of a red fluorescent gold nanocluster taking transferrin as a template, which comprises the following steps:

respectively preparing HAuCl₄And Tf in a molar ratio of 20-60:1, stirring at room temperature for 2-5 min, adjusting the pH of the mixed solution to 12 with NaOH solution, placing the solution in a microwave reactor, sealing, and reacting at 70-90 ℃ for at least 50min to obtain AuNCs @ Tf with strong red fluorescence emission. Then the mixture is put into a refrigerator with the temperature of 4 ℃ for standby.

The HAuCl₄And Tf is preferably 40: 1.

The reaction condition in the microwave reactor is preferably 60min at 80 ℃.

The prepared red fluorescent gold nanocluster can be applied to detection of copper ions and glutathione and also can be applied to targeted lysosome identification of cancer cells and encryption and decryption of fingerprint information.

Compared with the prior art, the invention has the beneficial effects that: the fluorescent gold nanoclusters are good in biocompatibility, strong in fluorescence, green in reaction conditions and free of pollution; compared with the previous research, the transferrin used in the method is low in proportion and short in reaction time, and the detection of copper ions is visualized by manufacturing copper ion test paper; AuNCs @ Tf-Cu due to non-luminescence²⁺AuNCs @ Tf was released under the action of GSH, which was much higher in tumor cells than in normal cells, and fluorescence was restored. Using this property, quenched AuNCs @ Tf-Cu was²⁺The normal cells and the cancer cells are distinguished by adding the normal cells and the cancer cells, respectively, and only the fluorescence of the cancer cells is recovered, but the normal cells are unchanged. In addition, the fluorescence in cancer cells alone is also evidence for the present invention by culturing normal cells and cancer cells in combinationThe gold nanoclusters have the recognition property on cancer cells, and a simple and feasible method is provided for early diagnosis of cancer.

Drawings

FIG. 1 is a graph of the effect of different preparation conditions on AuNCs @ Tf; wherein: A. the influence of the reaction molar ratio on AuNCs @ Tf, the influence of B and reaction time on AuNCs @ Tf, the influence of C and reaction temperature on AuNCs @ Tf, and the influence of D and reaction pH on AuNCs @ Tf.

FIG. 2 is a representation of AuNCs @ Tf; wherein: A. ultraviolet and fluorescence spectra of AuNCs @ Tf, infrared analysis spectrum of B, AuNCs @ Tf, transmission electron microscopy analysis spectrum of C, AuNCs @ Tf (particle size analysis map of AuNCs @ Tf in inset), X-ray diffraction analysis spectrum of D, AuNCs @ Tf, photoelectron spectrum analysis spectrum of E, X ray, and X-ray photoelectron spectrum analysis spectrum of F, Au 4 f.

FIG. 3 shows the selectivity and sensitivity of AuNCs @ Tf to copper ions; wherein A, AuNCs @ Tf and each metal ion interaction relative fluorescence comparison diagram, B, the fluorescence emission spectrum of AuNCs @ Tf added with copper ions of different concentrations, C, the linear relation diagram of copper ion concentration and fluorescence intensity, D, AuNCs @ Tf test paper under ultraviolet lamp and copper ion action diagram of different concentrations.

FIG. 4 is AuNCs @ Tf-Cu²⁺Selectivity and sensitivity to GSH; wherein A, GSH of different concentrations are added into AuNCs @ Tf-Cu²⁺The fluorescence emission spectrum of (1), a linear relation graph of the concentration of B and glutathione to the fluorescence intensity, C, AuNCs @ Tf-Cu²⁺Graph comparing relative fluorescence with biomolecular and ionic interactions for GSH (150. mu.M) in the presence of D, homocysteine (Hcy, 15. mu.M) and cysteine (Cys, 15. mu.M) at AuNCs @ Tf-Cu²⁺The fluorescence intensity of (1).

FIG. 5 Effect of different concentrations of AuNCs @ Tf on human cervical cancer cell (HeLa) activity.

FIG. 6 is a diagram of co-localization of AuNCs @ Tf to lysosomes as observed by confocal laser microscopy.

FIG. 7AuNCs @ Tf-Cu²⁺For GSH sensing and cancer cell recognition in lysosomes; wherein A, AuNCs @ Tf-Cu is observed by a fluorescence microscope²⁺With human cervical cancer cells (HeLa), human liver cancer cells (HepG2), human colon cancer cells (HCT116) and small cellsMurine fibroblasts (3T3) acted with different time profiles, B, AuNCs @ Tf-Cu²⁺Relative fluorescence intensity plots with different cell incubation times (data from FIG. 7A), C, AuNCs @ Tf-Cu²⁺Human cervical cancer cells (HeLa) and mouse fibroblasts (3T3) which enter different cells and have relative rate constants of D and the number ratio of 1:1 are mixed and cultured, and AuNCs @ Tf-Cu is added²⁺The image was observed after 1h with a fluorescence microscope.

FIG. 8 graph of AuNCs @ Tf vs. different cell effects; A. the relative rate constants of AuNCs @ Tf into different cells, B, AuNCs @ Tf versus the relative fluorescence intensity of different cell incubation times (data from FIG. 8A), C, AuNCs @ Tf versus time, were observed with a fluorescence microscope for the effects of AuNCs @ Tf on human cervical cancer cells (HeLa), human liver cancer cells (HepG2), human colon cancer cells (HCT116), and mouse fibroblasts (3T 3).

FIG. 9 AuNCs @ Tf on Filter paper Cu was added sequentially²⁺And photographs and fluorescence images of GSH; wherein, A, Chinese characters, B, fingerprint.

Detailed Description

The materials used in the examples are as follows:

transferrin (halo-Transferrin human, molecular weight 7.7X 10⁴) Manufactured by Sigma reagent limited;

tetrachloroauric acid (HAuCl)₄·4H₂O, molecular weight 411.9) produced by Shanghai Yangyun chemical reagent factory;

sodium hydroxide (NaOH, molecular weight 40.0) is produced in Beijing chemical plant;

glutathione (C)₁₀H₁₇N₃O₆S, molecular weight is 307.33) for production of industrial organisms;

various biomolecules, small molecules and ions (cysteine Cys, homocysteine Hcy, phenylalanine PHE, lysine Lys, tyrosine Tyr, glucose, ascorbic acid VC, potassium ion K⁺Sodium ion Na⁺Calcium ion Ca²⁺Sulfur ion S^2-Iodide ion I^-Oxalate ion C₂O₄ ^2-Cyanide ion CN^-)

Tris (hydroxymethyl) aminomethane (b)C₄H₁₁NO₃Molecular weight 121.14) was produced by the institute of optometry and fine chemistry in Tianjin.

Example 1

And (3) reducing and coating the transferrin to prepare the red fluorescent gold nanocluster.

5mg of transferrin (Tf) was weighed and lmL double distilled water was added to prepare a 5mg/mL transferrin solution. 206.0mg of tetrachloroauric acid (HAuCl) was weighed out₄·4H₂O), 0.01M HAuCl was prepared in a 50mL brown volumetric flask₄The solution was stored in a refrigerator at 4 ℃ until use. 10mL of a 1M NaOH solution was prepared. 1mL of 0.01M HAuCl was taken₄The solution was diluted four times with bidistilled water. 1mL of the diluted solution was placed in a microwave tube, and 1mL of the transferrin solution was added thereto and stirred at room temperature for 5 minutes. After the pH of the mixed solution was adjusted to 12 with 40. mu.L of a 1M NaOH solution, the solution was placed in a microwave reactor and sealed. The reaction conditions were set at 80 ℃, 60min, 150W, 250psi, high stirring speed, and pre-stirring time of 2 min. After the reaction, the reaction mixture was placed in a refrigerator at 4 ℃ for further use (FIG. 1 shows the change of HAuCl in the preparation of the material₄And Tf mole ratio, temperature, time, pH to determine optimal reaction conditions).

Example 2

(1) And (3) ultraviolet characterization and fluorescence characterization of the gold nanoclusters AuNCs @ Tf.

As observed in the inset of fig. 2A, the synthesized material appears brown under fluorescent light and has intense red fluorescence under uv light. 200 mu L of the prepared AuNCs @ Tf solution is added into 800 mu L of double distilled water for dilution, and an ultraviolet absorption curve and a fluorescence emission curve are determined. The ultraviolet absorption result shows that the absorption is obvious at 280nm, and the fluorescence spectrum shows that the optimal emission peak is 640 nm.

(2) And (3) infrared analysis (FTIR) spectrum characterization of the gold nanoclusters AuNCs @ Tf.

To confirm the binding of transferrin to the AuNCs @ Tf surface, the AuNCs @ Tf liquid produced was freeze-dried to a solid. Mixing transferrin and potassium bromide according to the mass ratio of 1:100, grinding and tabletting respectively, and measuring the infrared spectrum of the mixture; then, the prepared AuNCs @ Tf and potassium bromide are mixed, ground and tableted according to the mass ratio of 1:100, and the infrared spectrum of the mixture is measured.

In fig. 2B, their spectra are almost indistinguishable, indicating that transferrin has been immobilized on the surface of AuNCs. The stretching vibration of transferrin, C ═ O and N-H, was 1654cm^-1And 1540cm^-1Whereas the peak of AuNCs @ Tf shifts, this transition is mainly due to the change in the transferrin backbone structure caused by the interaction of Tf and Au. AuNCs @ Tf at 881cm^-1And 798cm^-1A new peak is shown, which may be attributed to the quinone-like unsaturated carbon-carbon double bond. This is probably due to the fact that the tyrosine residue (enol structure) in transferrin is replaced by HAuCl₄Oxidized to quinone.

(3) And (3) a transmission electron microscope analysis spectrogram and a particle size analysis spectrogram of the gold nanoclusters AuNCs @ Tf are represented.

In order to confirm the morphology and size of the gold nanoclusters, the AuNCs @ Tf solution is ultrasonically dropped on a copper net for half an hour to prepare a sample, and the liquid is observed by a transmission electron microscope after being volatilized. The sonicated AuNCs @ Tf liquid was also placed in a Malvern particle sizer to measure particle size.

FIG. 2C is a transmission electron microscopy analysis chart of the AuNCs @ Tf, which shows that the AuNCs @ Tf is uniformly dispersed and spherical, and the particle size is 4.75 + -0.5 nm. The inset is a particle size analysis spectrogram of the prepared AuNCs @ Tf, and the result shows that the hydraulic diameter is 6.25 +/-0.7 nm and is consistent with the result of a lens electron microscope.

(4) And (3) carrying out X-ray diffraction analysis (XRD) spectrum characterization on the gold nanoclusters AuNCs @ Tf.

In order to observe the crystal form state of the prepared AuNCs @ Tf, a prepared AuNCs @ Tf liquid sample was dried by a freeze dryer to obtain a solid, and the solid was ground to prepare a sample for measurement.

In fig. 2D, the characteristic diffraction peaks for AuNCs @ Tf are 31.5 °, 45.2 °, 68.7 °, and 75.2 °, respectively, corresponding to lattice planes (111), (200), (220), and (311), respectively. This indicates that AuNCs @ Tf is in a face centered cubic (fcc) structure.

(5) And (3) carrying out X-ray photoelectron spectroscopy (XPS) spectrum characterization on the gold nanoclusters AuNCs @ Tf.

To identify the functional groups and elements on the AuNCs @ Tf surface, the prepared liquid samples were freeze-dried to give solids and characterized on an X-ray photoelectron spectrometer.

As shown in FIG. 2E, there are five elements Au, S, O, C and N on AuNCs @ Tf. XPS spectra of Au 4F spectra as shown in FIG. 2F confirmed the presence of two different Au 4F spectra_7/2The doublets, one at 84.4eV and the other at 87.8eV, were assigned to Au (0) and Au (I), respectively. For AuNCs @ Tf, the ratio of Au (1)/Au (0) is 1.51, which means that Au (I) (the predominant form of Au-S bond) is 60.2% of all Au atoms in AuNCs @ Tf. It is generally believed that aggregation of the au (i) -thiolate complex is the primary cause of gold nanocluster (AuNCs) luminescence.

Example 3

Fluorescence studies of the interaction of various ions with the synthesized gold nanoclusters AuNCs @ Tf.

2mL (Mg) of each ionic solution of 0.1M was prepared²⁺,Pb²⁺,K⁺,Cd²⁺,Cr³⁺,Bi³⁺,Zn²⁺,Na⁺,Al³⁺,Ca²⁺,Ag⁺,Mn²⁺, Ba²⁺,Cu²⁺,Hg²⁺,Co²⁺,Ni²⁺) In a 2mL EP tube, 1mL of the prepared AuNCs @ Tf solution was diluted with 9mL of a Tris-HCl solution having a pH of 4.5. Setting the parameters (lambda) of the fluorescence spectrometer_ex＝410nm,λ_em570-740 nm), taking 1mL, placing in a fluorescence cuvette, scanning the sample, and recording data; add 10. mu.L of Mg to the cuvette²⁺The solution was stirred and timed for 2min, the sample scanned, the data recorded, and the experiment repeated with other ions. FIG. 3A records the results of the experiment, which demonstrate that Cu²⁺Can quench the fluorescence of AuNCs @ Tf.

Example 4

Fluorescence study of the interaction of copper ions with the synthesized gold nanoclusters AuNCs @ Tf.

Preparing Cu with different concentrations²⁺And (3) solution. 1mL of the prepared AuNCs @ Tf solution was diluted with 9mL of a Tris-HCl solution (pH 4.5). Setting the parameters (lambda) of the fluorescence spectrometer_ex＝410nm,λ_em570nm-740nm), 1mL is put into a fluorescence cuvette to scan the sampleRecording data; add 10. mu.L of Cu to the cuvette²⁺Stirring the solution, timing for 2min, scanning the sample, recording data, and adding Cu with different concentrations²⁺The above experiment was repeated with the solution. The results in FIGS. 3B and 3C show that Cu is present in a range of concentrations (0-2300nM)²⁺The concentration of (a) was well linear with respect to the relative fluorescence intensity, and the detection limit was 232 nM.

Example 5

Cu²⁺And (5) manufacturing the fluorescent test paper.

Soaking AuNCs @ Tf in filter paper and drying, soaking the filter paper in copper ions (10 μ M,20 μ M,40 μ M, 100 μ M,200 μ M,400 μ M) with different concentrations, drying, exciting with 365nm ultraviolet lamp, and obtaining Cu with different concentrations as shown in FIG. 3D²⁺After interaction with AuNCs @ Tf, AuNCs @ Tf-Cu²⁺Change in fluorescence of Cu²⁺The measurement of (2) is visualized.

Example 6

Glutathione and AuNCs @ Tf-Cu²⁺Fluorescence study of the interaction of the system.

Glutathione solutions with different concentrations are prepared. 1mL of the prepared AuNCs @ Tf solution was diluted with 9mL of a Tris-HCl solution having a pH of 4.5, and 100. mu.L of 0.04M Cu was added thereto²⁺The solution was stirred. Setting the parameters (lambda) of the fluorescence spectrometer_ex＝410nm,λ_em570-740 nm), placing 1mL of the solution in a fluorescence cuvette, scanning the sample, and recording data; adding 10 mu L of glutathione solution into a fluorescent cup, stirring, timing for 2min, scanning a sample, and recording data; the above experiment was repeated with different concentrations of glutathione solution. The results in FIGS. 4A and 4B show that the concentration of glutathione has a good linear relationship with the relative fluorescence intensity in a certain concentration range (0-150. mu.M), and the detection limit is 2.86. mu.M.

Example 7

AuNCs@Tf-Cu²⁺Selectivity of the system to GSH.

2mL of various biomolecules, small molecules and ionic solutions (cysteine Cys, homocysteine Hcy, phenylalanine PHE, lysine Lys, tyrosine Tyr, glucose, and ascorbic acid) of 0.01M are preparedAscorbic acid, potassium ion K⁺Sodium ion Na⁺Calcium ion Ca²⁺Sulfur ion S^2-Iodide ion I^-Oxalate ion C₂O₄ ^2-Cyanide ion CN^-). 1mL of the prepared AuNCs @ Tf solution was diluted with 9mL of a Tris-HCl solution having a pH of 4.5, and 100. mu.L of 0.04M Cu was added thereto²⁺The solution was stirred. Setting the parameters (lambda) of the fluorescence spectrometer_ex＝410nm,λ_em570-740 nm), placing 1mL of the solution in a fluorescence cuvette, scanning the sample, and recording data; adding 10 mu L of cysteine solution into the fluorescent cup, stirring, timing for 2min, scanning the sample, and recording data; the titration was repeated until the fluorescence no longer changed and the data was saved. Other small biological molecules the above experiment was repeated. FIG. 4C records the results of the experiment, which demonstrate AuNCs @ Tf-Cu²⁺Has selectivity to glutathione.

Furthermore, we detected AuNCs @ Tf-Cu since intracellular GSH content was 10 times that of Cys²⁺Effect on GSH detection in the presence of other biological thiols. Placing 1mL of the quenched solution in a fluorescent cuvette to scan a sample, and recording data; adding 10 mu L of 15.3mM glutathione solution and 10 mu L of 1.53mM homocysteine solution into a fluorescent cup, stirring, timing for 2min, scanning a sample, and recording data; 1mL of the quenched solution was placed in a fluorescence cuvette, 10. mu.L of a 15.3mM glutathione solution and 10. mu.L of a 1.53mM cysteine solution were added to the cuvette, and after stirring, the time was kept for 2min, and the sample was scanned and the data was recorded. As shown in fig. 4D, the presence of Hcy (0.1 equivalent) or Cys (0.1 equivalent) did not interfere with the detection of GSH. This suggests AuNCs @ Tf-Cu²⁺Selective detection of GSH.

Example 8

Study of cytotoxicity experiments.

Human cervical cancer cells (HeLa) (10% FBS/DMEM medium, 5% CO) in logarithmic growth phase₂37 ℃ C.) at 5X 10 per well³Inoculating to 96-well culture plate, and changing to 200 μ L culture plate containing AuNCs @ Tf (concentration from large to small: 0 μ M, 50 μ M, 100 μ M,200 μ M,400 μ M, 600 μ M) after cell adherenceAfter culturing for 24h, removing the culture solution containing AuNCs @ Tf, adding 200 mu L of fresh culture medium into each well, adding 20 mu L of 5mg/ml MTT solution into each well, continuously culturing for 4h, removing the old culture solution from each well, adding 150 mu L of LDMSO into each well, shaking for 10min, and performing MTT detection on a microplate reader.

FIG. 5 is a graph of the effect of AuNCs @ Tf at various concentrations on human cervical cancer cell (HeLa) activity. As can be seen, the survival rate of the cells was more than 80% even after incubation with high concentrations of AuNCs @ Tf, indicating that the material had relatively low toxicity.

Example 9

Distribution of AuNCs @ Tf within cells.

To further determine the location of AuNCs @ Tf in cells, a green lysosomal probe was used. HeLa cells were cultured at a temperature of 1.0X 10⁵The density of each cell was plated on a 35mm dish, and AuNCs @ Tf (1mg/mL) was added to the dish after 20 hours of culture. After 1 hour, wash 3 times with PBS to remove free AuNCs @ Tf. After a green lysosomal probe was then added to the cells and incubation continued for 30 minutes, it was washed 3 times to remove free lysosomal probe. Finally, cells were observed with a confocal laser microscope. AuNCs @ Tf was excited at 405nm and the emission was collected at 600-700 nm. The green lysosomal probe was excited at 488nm and emission was collected at 500-600 nm.

As shown in fig. 6A, AuNCs @ Tf was observed to fluoresce red, LysoTracker was observed to fluoresce green, and the composite image was observed to fluoresce orange, indicating AuNCs @ Tf and lysosomal localization. In general, the co-localization index is good when the co-localization coefficient is close to or greater than 0.5 (r.gtoreq.0.5). Therefore, we can be said that AuNCs @ Tf has entered lysosomes (FIG. 6B).

Example 10

Identification of tumor cells.

Human cervical cancer cells (HeLa), human liver cancer cells (HepG2), human colon cancer cells (HCT116) and mouse fibroblasts (3T3) were combined at 1.0X 10⁵The density of each cell was inoculated into a 35mm dish (the first three represent tumor cells and the latter normal cells), and 20 hours after the culture, AuNCs @ Tf-Cu²⁺(Off) the system was added to a petri dish and photographed with a fluorescence microscope at different times. In the experiment, AuNCs @ Tf (on) was chosen as a control.

To further illustrate AuNCS @ Tf-Cu²⁺Ability to identify tumor cells, HeLa cells and 3T3 cells were mixed in a number to volume ratio of 1: 1. After the cells were adherent, 2mg/mL AuNCs @ Tf-Cu was added²⁺Add to the dish for 1 hour and take pictures with a fluorescence microscope.

As shown in FIG. 7A, HeLa, HepG2 and HCT116 cells were compared to AuNCs @ Tf-Cu²⁺Incubated together, initially without fluorescence, and with time the red fluorescence signal gradually appeared and became bright. Furthermore, we found that of the three cancer cells in FIG. 7B, the enhancement of fluorescence intensity by GSH to HepG2 cells was brightest, with pseudo-first order rate constants enhanced by 2-fold and 5-fold compared to HeLa and HCT116 cells (FIG. 7C, Table 1), suggesting AuNCs @ Tf-Cu²⁺The sensitivity and selectivity of the system to HepG2 cells was optimal. While normal cells (3T3) were associated with AuNCs @ Tf-Cu²⁺When incubated together, no fluorescence was observed even when the incubation time reached 2 hours (fig. 7A). As a control, bright fluorescence was also observed after 2 hours incubation of AuNCs @ Tf and 3T3 cells (fig. 8A). This phenomenon was also found in three cancer cells, and the rate of uptake of AuNCs @ Tf by HepG2 cells was the fastest among the three cancer cells (fig. 8B, 8C). This may explain the AuNCs @ Tf-Cu uptake by HepG2 cells²⁺Quickly results in a fast response of the GSH to the system.

To further illustrate AuNCs @ Tf-Cu²⁺For GSH selectivity, we mixed HeLa cells and 3T3 cells into culture dishes in a quantitative ratio of 1: 1. After the cells were adherent, AuNCs @ Tf-Cu was added²⁺Add to the petri dish. After 1 hour, the cells were observed. As shown in fig. 7D, there was red fluorescence in the cancer cells marked in the circles, while no fluorescence was observed in the normal cells. The interesting result shows that the gold nanocluster material can target lysosomes to recover the fluorescence of cancer cells in situ, has wide prospect in the aspect of identifying the cancer cells, and can be applied to diagnosis and prevention of cancers.

TABLE 1

Example 11

AuNCs @ Tf was studied as a way to encrypt and decrypt information.

We filled the fountain of AuNCs @ Tf with Chinese characters on filter paper in a pen, and dried in air. Then irradiated with 365nm UV lamp. Then we will put Cu²⁺The solution was dropped on the Chinese character and irradiated with an ultraviolet lamp. Finally, the GSH solution was dropped on the chinese characters and irradiated with an ultraviolet lamp.

We dipped AuNCs @ Tf in the fingers and printed the fingerprint on filter paper, dried in air. The fingerprint was then illuminated using a 365nm UV lamp. Then we will put Cu²⁺The solution was dropped on the fingerprint and irradiated with an ultraviolet lamp. Finally, the GSH solution was dropped on the fingerprint and irradiated with an ultraviolet lamp.

Based on Cu²⁺And the stimulus response of GSH to fluorescent signals, we have devised a novel information encryption and decryption scheme. We written Chinese characters and printed fingerprints with AuNCs @ Tf on filter paper (FIG. 9), and when exposed to 365nm UV light, the Chinese character and fingerprint information was clearly visible. Mixing Cu²⁺After loading on filter paper impregnated with AuNCs @ Tf, the fluorescence of the Chinese characters and fingerprints was quenched, thus we encrypted the information. We then loaded GSH into the impregnated AuNCs @ Tf-Cu²⁺On the Chinese characters and fingerprints. The quenched fluorescence is recovered and we can retrieve the information again. This phenomenon shows that AuNCs @ Tf also has great application prospect in the information encryption and decryption processes.

Claims (3)

1. An application of a red fluorescent gold nanocluster taking transferrin as a template in encryption and decryption of fingerprint information is characterized in that the preparation method of the gold nanocluster comprises the following steps: respectively preparing HAuCl₄ And an aqueous solution of transferrin (Tf) in a molar ratio of 20-60:1, stirring at room temperature for 2-5 min, adjusting the pH of the mixed solution to 12 with NaOH solution, placing the solution in a microwave reactor, sealing, and reacting at 70-90 deg.CAuNCs @ Tf should be obtained with a strong red fluorescence emission for at least 50 min.

2. The use according to claim 1, wherein the HAuCl is₄ And Tf was 40:1 molar ratio.

3. The use according to claim 1, wherein the reaction conditions in the microwave reactor are 80 ℃ for 60 min.

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